1.

0 OBJECTIVE
1.1

To study the media development for yeast fermentation in a bioreactor.

1.2

To know the criteria and identify the areas of sterilization before run the
experiment.

2.0 INTRODUCTION
Fermentation is a metabolic process in which bacteria, yeast or other
microorganisms break down organic matter to obtain the energy required to stay alive,
and produce organic compounds such as alcohol and organic acid, as well as inorganic
compounds such as carbon dioxide and hydrogen. Fermentation also refer to the use
of yeast to change sugar into alcohol or the use of bacteria to create lactic acid in
certain foods. Fermentation is widely applied in industry for the production of wine,
mead, cheese and beer.
All fermentation process required media for the microorganism to growth. A
media must have sufficient nutrient to ensure the microorganism grow in an optimum
condition to have high yield of desired products. Sterilization is an important
technique in fermentation to prevent the contamination by killing the unwanted
microorganism that can affect the product in growth media. Hence, the bioreactor
must be autoclaved before the fermentation process start. In order to prepare a suitable
medium for the growth of microbes, the main factors to be considered is the sources
of carbon and nitrogen as well as vitamins or any other limiting elements and minor
minerals which will affect the growth rate of cell.
Bioreactor is a vessel where a chemical process which involves organisms or
biochemically active substances is carried out. The microorganisms or cells are able to
perform their desired function with limited production of impurities under optimum
condition. The environmental conditions inside the bioreactor, such as temperature,
nutrient concentrations, pH, and dissolved gases (especially oxygen for aerobic
fermentations) affect the growth and productivity of the organisms. A properly
designed bioreactor is used to provide a controlled environment to achieve optimal
growth and product formation in the particular cell system employed.

3.0 PROCEDURES

5.0 QUESTIONS
5.1 List down the factors that can cause contamination to your culture.
If we do not sterilize or autoclave our bioreactor and glassware (like tubes),
contamination is easier to occur. Besides, as fermentation occurs, inhibitory
compound will be produced which then will mix with the products causing
contamination and low yield of the products. Poor aseptic techniques also cause
contamination. Improper handling can contaminate the cell line, but this infection
is likely to be detected and controlled before inoculation of the bioreactor. Cell
culture fermentation is susceptible to microbial contamination due to a longer
fermentation process and slower growth rate.
5.2 Why pH probe needs to be calibrating before autoclave start and pO 2 probe after
autoclave?
Before the start of autoclaving of the broth for any cultivation experiment, it is
essential to calibrate the pH probe. The measured signal is compared with the set
point in the controller unit which then activates the acid or alkali to bring the
measured value close to the set point. However before the pH probe is used, it
needs to be calibrated with two buffers usually in the pH range which is to be
used in the bioreactor cultivation experiment. This is because only a narrow pH
ranges to culture the cell. For this specific pH controller one has to suitably
identify the right control action setting for the addition of certain concentration of
acid / alkali in the desired fermentation broth which can give quick control action
with-out any oscillations/offset of measured value around the set point.
5.3 What are the differences between impeller design for microbial, mammalian, and
plant bioreactor and why they are designed in different ways?

Microbial Cell (Yeast

Mammalian Cells Bioreactor

Plant Cells Bioreactor

and bacteria) Bioreactor

Rushton turbine is the Pitch

blade

impellers. Large

pitch

blade

most common generic Pitched-blade impellers are impeller.

term applied to flat- low shear impellers designed
bladed or disk-turbine to gently mix the contents of

Large, slow-moving axial

impellers Their blades a culture without causing cell flow impellers providing
are flat and set vertically damage. They are most often good mixing at relatively
along an agitation shaft, used
which

produces

unidirectional
flow.

or

mammalian, low tip speeds up to 2.5 m

other

shear- sec-1 are superior for plant

radial sensitive cell lines growing cell and tissue cultures.

Rushton

Rushton-type

a insect,

with

and in

suspension.

These Axial flow impellers are

impellers impellers are often used in commonly used for more

are commonly used in batch or fed-batch cultures, sensitive

fermentations
lines

that

of
are

considered
sensitive,
yeasts,

cell but they can also be used for requiring

not continuous

and

lower

perfusion dispersion. There

shear processes.
including

bacteria,

cultures

and

some fungi.

gas
is

tendency to develop shear

sensitive improved axial
flow

impellers

characterized by profiled
large stirring paddles.

Impellers are designed in different patterns because different cells (animal, bacteria or
plant) have different cell structures. Therefore, different designs are created to suit the
different cell structures. In comparison to microbial cultures, cell cultures which are
not protected by a cell wall are much more sensitive to shear stress and foam
formation. Therefore for sensitive cell, low shear stress should be achieved by using
different impeller which is more flat to avoid cell disruption.

5.4 Why microsparger is being used in mammalian bioreactor?

Microsparger which normally have micro-sized (2-30m) pores for aeration and
they come from different size and shape. These spargers are made up of sintered
metals and sold as cylindrical elements to be connected to a main sparger line.
Microsparger line generate very small bubbles (100-400 m), which are
extremely efficient for mass transfer of oxygen. Small bubbles give high surface
area, for culturing mammalian cell which is animal cell, high oxygen level is
needed. Therefore, microsparger is more suitable to be used.

5.5 Give some comment on design for sparger, impeller, baffle and heating element
for these three types of bioreactor.
To augment mixing and gas dispersion, baffles are employed. They are normally
incorporated into agitated vessels of all sizes to prevent vortex and to improve
aeration efficiency. Baffles are metal strips roughly one-tenth of vessel diameter
and attached radially to the wall of bioreactor. Baffles should be installed in such
a way that a gap exist between them and vessel wall, so that there is scouring
acting around and behind the baffles thus minimizing microbial growth on the
baffles and fermenter walls.
Description

Sparger

Microbial Cell

Mammalian Cells

Plant Cells

(Yeast and bacteria)

Bioreactor

Bioreactor

Bioreactor
Microsparger (2- Macrosparger (also Macrosparger (also
30m)

known

Impeller

Rushtan Turbine

sparger,0.5-2mm)
sparger,0.5-2mm
Large pitch blade Pitch blade disk

Baffle

Adding

impeller
baffles Adding

as

ring known

as

ring

turbine
baffles With animal cell

prevent vortex.

culture

baffles

Generally four to Generally four to causes

shear

eight

prevent vortex.

baffles

are eight

incorporated

baffles

incorporated

are damage, instead of

baffles

bottom

drive

axial

impellers

slightly

off sight of centre

Heating element

Temperature

Temperature

is used.
Temperature

maintained at 300C maintained at 25- maintained at 29by water jacket to 270C

maintain

by

its jacket.

water 370C

by

heat

blanket.

temperature.

5.6 How much yeast biomass will be produced theoretically from the stoichiometric
equation above?
C6H12O6 + 3O2 + 0.48NH3

0.48C6H10NO3 + 4.32H2O + 3.12CO2

Yeast biomass = 0.48[12(6) + 1(10) + 14 + 16(3)]

= 69.12
Therefore, the yeast biomass will be produced theoretically from the
stoichiometric equation is 69.12g dry mass/ g glucose consumed

5.7

Determine

the

yield

coefficients

Yx/s

(biomass/glucose)

and

Yx/o2

(biomass/oxygen).

Yx/s =

0.48C 6 H 10 NO 3
C 6 H 12O 6

0.384 g biomass/g glucose

0.48[12 ( 6 )+10+14 +16 ( 3 ) ]

12 ( 6 )+ 12+16 (6)

69.12
180

Yx/o2 =

0.48C 6 H 10 NO 3
3O 2

69.12
32

= 2.16 g biomass/g oxygen

5.8 What will happen if you have mistakenly tighten the lid screw and not clipped the
hose for aeration during sterilization?
If the lids screw is tighten before sterilization, the steam inside the vessel will
explode since high temperature will cause pressure inside to be higher. Therefore,
if the pressure inside the vessels is too high, the steam explosion maybe will
cause the vessel to be broken. Besides, the hose normally will be clipped and
covered with aluminium foil to be sterilized. If the hose is not clipped and left for
autoclave, the hose will easily be damaged and even the media inside the vessels
will flow out.

6.0 DISCUSSION
Bioreactor also known as fermenter, can be described as a vessel which has
provision of cell cultivation under sterile condition and control of environmental
conditions such as pH, temperature and dissolved oxygen. It can be used for the
cultivation of microbial plant or animal cells. A typical bioreactor consists of
following parts: agitator, motor, stirrers, jackets and baffle. Agitator facilitates the
mixing of the contents of the reactor which eventually keeps the cells in the perfect
homogenous condition for better transport of nutrients and oxygen for adequate
metabolisms of cell to the desired products. The agitator can be driven by magnetic
/mechanical agitators. Baffle in the reactor is to break the vortex formation in the
vessel, which is usually highly undesirable as it changes the centre of gravity of the
system and consumes additional power. Sparger is to supply oxygen to the growing
cells. Bubbling of air through the sparger not only provide the adequate oxygen to the
growing cells but also helps in the mixing of the reactor contents thereby reducing the
power consumed to achieve a particular level of (mixing) homogeneity in the culture.
Jacket keeps the temperature of the bioreactor at a constant value. The desired
temperature of the circulating water is maintained in a separate Chilled Water
Circulator. The contact area of jacket provides adequate heat transfer area wherein
desired temperature water is constantly circulated to maintain a particular temperature
in the bioreactor.
In this experiment, we are going to explore with three different types of
bioreactor that are plant, mammalian and yeast/bacterial cells. There are some
controllers in the bioreactor that are used to control parameter. Temperature, media
flow rate, stirrer speed, pO2 detector, pH, antifoaming detectors, level control and gas
flow rate(air).If there are any parameter that changes during process, that particular

controller will be detected and action will be taken to return the parameter back to
normal(set value).For oxygen detector, if the oxygen probe detect there are low
concentration of oxygen level in the vessel, controller will take action by injecting
more oxygen to the vessel. For the oxygen probe, the bottom part is made up of
membrane which the oxygen will dissolve in it. Whole bioreactors have to be
autoclaved by covering with aluminium foil under 120 0C to kill all the contaminants.
Filter in the bioreactor are used to prevent the bacteria out of the bioreactor. The filter
got its own lifetime limits. Media can be autoclaved/sterilised while vitamin or
minerals cannot be sterilised as it will destroy vitamin itself. While for the yeast,
which occupied 10% of working volume is injected into the vessel after the
bioreactor has been autoclaved via the largest pore on the upper surface of the
bioreactor.
For the yeast or bacteria bioreactor, the tank volume capacity is up to 2L.There
are four closed tubes which are acid, alkali, media and antifoaming on the upper
surface of the reactor. The water jacket is used to maintain the temperature inside the
reactor. If we look outside from the reactor, there are 2 layers inside it, which one is
jacket and the others is vessel. Jacket temperature is greater than the temperature
inside the vessels. The impeller using in the yeast or bacteria reactor is rushtan
impeller which have different diameter and size compare to others. Impeller normally
contains 6 blades. Their blades are flat and set vertically along an agitation shaft,
which produces a unidirectional radial flow. Rushton and Rushton-type impellers are
commonly used in fermentations of cell lines that are not considered shear sensitive,
including yeasts, bacteria, and some fungi. Inside the reactor which contain level
detector and foam detector. Level detector is used to detect the volume of the media
culture to prevent overflow while the foam detector is to detect the foam. Baffle is
installed in the reactor to prevent vortex. Under the rushton impeller contain
microsparger. Microsparger produces small bubbles which gives high surface area to
provide more oxygen to the vessels .Besides, there are syringe connected to bioreactor which function as sucking out the samples in particular time.

While for plant cell cultures bioreactor, the tank volume capacity is bigger
than the yeast and mammalian cell. The tank volume is around 5L.There are also

some different design of impeller used which is large pitch blade impeller which is
suitable for plant (sensitive cell) to provide a low shear sensitivity. Ring spurge is
used which the holes is bigger than microsparger. The bubbles produced is bigger than
microsparger. The similarity between plant and yeast is both of them using water
jacket to maintain temperature constant.

On the other side, for mammalian cell, the size is same like yeasts bioreactor
which is 2L.The only difference compare to plant and microbial is mammalian cells
bioreactor using heat blanket instead of water jackets. The heat blanket is like the
jacket that covered the whole outside vessels. Ring spurge is used which the holes is
bigger than microsparger. The bubbles produced is bigger than microsparger. Besides,
the speciality of mammalian cells bioreactor compare to the rest two is the bioreactor
is put on the weight balance. This weight balance is used to detect and measure the
weight before and after the fermentation process.

7.0 CONCLUSION
8.0 REFERENCES
9.0 APPENDIX
9.1 Microbial Cell Bioreactor

9.2 Mammalian Cell Bioreactor

9.3 Plant Cell Bioreactor